Signaling through multisubunit immune receptors is regulated through intracellular tyrosine kinases. These pathways require macromolecular assemblies involving intracellular tyrosine activation motifs (ITAMs) on the receptors and kinases such as syk and fgr. This project aims at examining the order of assembly, the affinities and kinetics of the molecular steps in cell activation. A complete description is provided in the collaborative projects of the Year 16-20 NFCR renewal. In brief, ITAM or kinase reagents are being displayed on beads through avidin-biotin or GSH-GST linkages. The second components are fluorescently labeled and flow cytometry is used to detect and analyze the binding interactions. We have displayed phosphorylated ITAMs on beads and detected them with anti-phosphotyrosine antibodies. Current studies are aimed examining the activities of specific phosphatases active on these ITAMs that regulate intracellular signaling pathways.